Method and composition for increasing cardiac inflow

ABSTRACT

A pharmaceutical composition for treatment of persons afflicted of heart failure with preserved ejection fraction (HFPEF), diastolic heart failure (DHF) or diastolic dysfunction (DF), the composition comprising a therapeutically effective amount of a compound capable of specific binding to the relaxin receptor (RXFP1) present on fibroblasts, fibromyoblasts, endothelial cells, endocardial cells, and cardiomyocytes in the cardiac muscle to increase the heart&#39;s stroke volume at lower end-diastolic pressure.

CROSS REFERENCE TO RELATED APPELLATIONS

This is a divisional application of U.S. application Ser. No.15/313,517, filed on Nov. 22, 2016, which is a U.S. National Stage ofInternational Application No. PCT/EP2015/061554, filed on May 26, 2015,which was published in English under PCT Article 21(2), which in turnclaims the benefit of European patent application No. 14169711.0 filedon May 23, 2014; each of the prior applications is incorporated hereinin its entirety.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for treatinghuman subjects afflicted with heart failure.

BACKGROUND OF THE INVENTION

Heart failure is commonly classified based on which heart function orwhich side of the heart is most affected. Left-sided heart failuredescribes a failure of the left-sided pumping action by whichoxygen-rich blood from the lungs is moved through the left atrium intothe left ventricle and then out into the rest of the body. The termright-sided heart failure is used for a failure of the right-sidedpumping action which pumps blood that returns to the heart through theveins through the right atrium into the right ventricle and then backout into the lungs to have it replenished with oxygen. The symptoms ofheart failure are further distinguished whether they have developedquickly (acute heart failure) or gradually over time (chronic heartfailure). Congestive heart failure (CHF) describes the general conditionin which the heart cannot pump enough blood to meet the needs of thebody. As blood-flow out of the heart slows, blood returning to the heartthrough the veins backs up, causing congestion in the tissues. Typicalsigns thereof are swollen legs or ankles, tiredness, difficulties ofbreathing, pulmonary edema, respiratory distress. CHF may be caused bycoronary artery diseases, congenital and primary heart diseases,infections of the heart muscle (cardiomyopathy, endocarditis and/ormyocarditis), myocardial infarctions, high blood pressure, heart valvediseases. CHF also affects the function of the kidneys. The drugs usedin treatment of CHF are angiotensin inhibitors and vasodilators toexpand blood vessels and decrease resistance, beta blockers to improvethe function of the left ventricle, digitalis to increase the pumpingaction of the heart and diuretics for an elimination of excess salt andwater.

The present disclosure relates to heart failures with preserved ejectionfraction (HFPEF), also named heart failure with normal ejection fractionor simply diastolic heart failure (DHF), and more specifically to aHFPEF subgroup involving altered chemo-mechanical properties of theheart muscle proteins. Diastole is that phase of the cardiac cycle whenthe heart is not contracting to propel blood out (systole) to the body,brain and lungs but instead is relaxing and filling with incoming bloodthat is being returned from the body through the inferior vena cava(IVC) and through the superior vena cava (SVC). The HFPEF subgroup ofheart failures is therefore associated with a decline in diastolicperformance of the left ventricle of the heart. When the cardiac musclehas become stiff and lost its ability to relax the left ventricle is notreadily filled with blood following contraction and the cardiac outputbecomes either diminished or an elevated ventricular diastolic pressuredespite essentially normal end diastolic volume (EDV) is observed forcompensation. The HFPEF is often characterized histologically by ahypertrophy of cardiomyocytes, increased interstitial collagendeposition and calcium deposition within the myocardium which areassumed to lead collectively to decreased distensibility and compliance.The chemo-mechanical characteristics of the heart muscle proteins aswell as myocytes and the biophysics of the failing heart have not yetachieved clinical relevance.

There is no specific treatment of HFPEF available. When the chroniccondition is tolerable by the patient, the therapy may be directed ataggravating factors such as high blood pressure and diabetes. Diureticsare often given. The administration of calcium channel and/orangiotensin II receptor blocker drugs may be of benefit in reducingventricular stiffness in some cases but there is no favorable effect inmortality rates. A major complication is pulmonary edema the treatmentof which by diuretics is often challenging since the stiffened heart andvessels of the patients are very susceptible to hypotensive events aftersalt and water excretion. Thus, there are no means and tools fortreating persons afflicted of heart failure with preserved ejectionfraction (HFPEF). The prior art therefore represents a problem.

SUMMARY OF THE INVENTION

The present invention provides a pharmaceutical composition fortreatment of persons afflicted of chronic heart failure with preservedejection fraction (HFPEF) and stiffening of the heart muscle, thecomposition comprising a therapeutically effective amount of a compoundcapable of specific binding to the relaxin receptor (RXFP1) present onfibroblasts, fibromyoblasts, endothelial cells, endocardial cells, andcardiomyocytes in the cardiac muscle to increase heart compliance andstroke volume and to lower the end-diastolic pressure of the leftventricle. The provided pharmaceutical composition can particularly beuse and administered for treating the effects of a deficientphosphorylation of the cardiospecific titin, notably ahypophosphorylation of the cardiospecific titin N2B. The composition maycomprise human relaxin molecules or a pharmaceutically acceptablederivative or precursor thereof in admixture with a pharmaceuticallyacceptable adjuvant, carrier, diluent or excipient suitable forsubcutaneous or intravenous injection or oral application.

The pharmaceutical composition may be formulated as an emulsion(oil-in-water or water-in-oil) for delivery to the oral orgastrointestinal tract mucosa. The relaxin may be contained in adelivery vehicle selected from among a micelle, inverse micelle,liposome, cubosome and a mixture thereof. A preferred embodimentconcerns a composition wherein a mucoadhesive protein is associated withthe delivery vehicle via a chemical or physical bond so that thecomposition adsorbs to the mucosa or is retained on a mucosal surfacefor effecting systemic delivery of the relaxin.

Another embodiment of the present disclosure relates to a compositionwhich is for subcutaneous infusion of human relaxin at a rate in therange of 10 μg/kg/day to 1000 μg/kg/day or as oral application producingplasma concentrations equivalent to those achieved with sc. infusions.The relaxin is preferably administered at an infusion rate in the rangeof 30 μg/kg/day to 100 μg/kg/day.

The disclosure further provides a pharmaceutical composition fortreatment of a subject suffering from chronic diastolic heart failureand diagnosed of a phosphorylation deficit of the cardiospecific titin.The subject may be renally impaired, having creatinine clearance in therange of 30 to 75 mL/min/1.73 m2 estimated according to the MDRDformula. The subject afflicted of chronic diastolic heart failure may befurther hypertensive and/or suffering from diabetes and/or oxidativestress and/or inflammation, i.e. typical concomitant diseases andchronic conditions which lead to a deficient phosphorylation of thecardiospecific titin. Another aspect of the disclosure concerns a methodfor increasing cardiac inflow by administering to a patient exhibitingpathologically diminished cardiac inflow a therapeutically effectiveamount of a compound capable of specific binding to a relaxin receptorin the myocardium and lowering the heart's end-diastolic pressures andincreasing the heart's stroke volume.

The pharmaceutical composition for treatment of persons affected by achronic diastolic heart failure with preserved ejection fraction (HFPEF)caused by altered chemo-mechanical characteristics of the heart muscleproteins, notably titin, and more precisely by a hypophosphorylation oftitin, said composition comprises a therapeutically effective amount ofa compound capable of specific binding to relaxin receptor (RXFP1)present on fibroblasts, fibromyoblasts, endothelial cells andcardiomyocytes in the cardiac muscle to thereby increase the heart'sstroke volume at lower pressures.

The disclosure further provides as active relaxin agent or ingredientthe receptor binding core structure (minimal active structure) of humanrelaxin, or a pharmaceutically acceptable derivative or precursorthereof, in admixture with a pharmaceutically acceptable adjuvant,carrier, diluent or excipient suitable for parenteral administration. Ina further embodiment, the pharmaceutical composition may comprise asactive ingredient or agent at least one selected from the groupcomprising human relaxin-1, human relaxin-2, human relaxin-3 as well asanalogues or derivatives thereof. A most preferred embodiment containssynthetic relaxin molecules which are bioequivalent to human relaxin-2.The synthetic human relaxin-2 may be chemically synthesized.

As mentioned, the pharmaceutical composition may be for injection,preferably for intramuscular, subcutaneous or, most preferably, forintravenous injection and the dose for human relaxin-2 may be in therange of 1 μg/kg/day to 1000 μg/kg/day. A subcutaneous injection ispreferred for obtaining a slow release of the relaxin peptide,preferably human relaxin-2. An administration delivered directly to theveins through an intravenous drip may preferably be in the range from 5μg relaxin-2/kg/day to 100 μg/kg/day.

It is further contemplated to administer the relaxin agent in aformulation as also used for an oral or nasal delivery andadministration of insulin. Such a galenic formulation may comprise afunctionally active amount of a mucoadhesive protein, a relaxin compoundas described and, optionally, additional agents for delivery; and adelivery vehicle associated with the agents. The composition may beformulated as an emulsion (oil-in-water or water-in-oil) and fordelivery to the oral or gastrointestinal tract mucosa. The mucoadhesiveprotein may be from among immunoglobulins, albumins, mucin proteins andtransferrins. The mucoadhesive protein may be associated with thedelivery vehicle via a chemical or physical bond, so that thecomposition adsorbs to the mucosa or is retained on a mucosal surfacefor effecting systemic delivery of the agent. The delivery vehicle maybe selected from among a micelle, inverse micelle, liposome, cubosomeand a mixture thereof. Such kind of targeted emulsions and formulationshave been described in detail for example in EP 1 768 647 B1, U.S. Pat.No. 8,414,914. Exemplary formulations for an oral administration ofrelaxins have been described. W02003/047494A2, U.S. Pat. No. 5,444,041and WO 02/094221A1 are related to emulsion/microemulsion compositions,W096/37215 A1 is related to peptide water in oil emulsions,US2006/0210622A1 is related to surface modified particulatecompositions, WO 03/030865 A1, U.S. Pat. No. 5,206,219A and US2004/097410A1 are related to peptide compositions including e.g.surfactants and/or lipid components, US2006/0182771 A1 is related toself-emulsifying compositions and W02008/145730A1, WO 2008/145728A1 andMa Er-Li et al., Acta Pharmacologica Sinica, October 2006, Vol. 27 (10):1382-1388, are related to microemulsions or emulsion pre-concentrates.SMEDDS compositions are known to improve the solubility and oralbioavailability of polypeptides such as cyclosporine, insulin andrelaxin. However, the solubility and bioavailability of hydrophilicwater soluble polypeptides such as human insulin or relaxin in SMEDDSand SNEDDS may not always be optimal.

The pharmaceutical composition is preferably administered to a subjectafflicted by heart failure with preserved ejection fraction (HFPEF) anddiagnosed of at least one symptom which brings about a differentialchange in titin domain phosphorylation. The relaxin-induced changes intitin-domain phosphorylation have been observed to fine-tune passivemyocardial stiffness so that the diastolic function of the heart can berecovered.

The titin-domain phosphorylation affecting symptom may be at least oneof the non-limiting group comprising diabetes, high blood pressure,ischemia, arteriosclerotic vascular diseases or any one of the group ofdiseases causing oxidative stress and/or an undersupply of the heartwith oxygen and nutrients (e.g. smoking) and which result in an alteredtitin-domain phosphorylation in the failing myocardium. An alteredphosphorylation of titin (hypophosphorylation of titin N2B isoform) inthe failing myocardium can for example be detected by isolating titinN2B from serum, e.g. titin exon 49 fragment released from the failingmyocardium, and by determining its phosphorylation by mass spectrometry.

The disclosed pharmaceutical composition may be administered to patientshaving at least one of the group comprising diabetes, atherosclerosisand/or high blood pressure and at high risk developing a heart failurewith preserved ejection fraction (HFPEF) due to altered chemo-mechanicalcharacteristics of the myocardium. In a most preferred embodiment, thepharmaceutical composition is administered to a subject afflicted of DHFor HFPEF who is also renally impaired. This is for example the case,when the subject has a creatinine clearance in the range of 30 to 75mL/min/1.73 m2. When the subject afflicted of DF, DHF or HFPEF isfurther hypertensive, the disclosed active ingredient may beadministered as described but preferably in a pharmaceutical compositionfurther comprising an antihypertensive drug. The antihypertensive drugmay be selected from the group of anti-hypertensive drugs comprisingvasodilators, adrenergic blockers, centrally acting alpha-agonists,angiotensin-converting enzyme inhibitors, angiotensin II receptorblockers, calcium channel blockers and diuretics.

Another aspect relates to a method for increasing cardiac inflow byadministering to a patient exhibiting pathologically diminished cardiacinflow of a therapeutically effective amount of a compound capable ofspecific binding to a relaxin receptor in the myocardium and increasingthe heart's stroke volume when the patient further exhibits either anon-typical expression of titin N2B, e.g. a shift in the expression ofthe titin isoforms N2B and N2A, or a deficient or hypo-phosphorylationof the cardiac protein titin N2B or both. The present invention shallhowever not be construed to be limited to a specific misphosphorylationof titin but relates to the posttranslational modifications of titin ingeneral. It should be pointed out that the primary physiological effectof relaxin in the treatment of heart failure with preserved ejectionfraction is the relaxin-induced change of the chemo-mechanicalproperties of the myocardium and an increased phosphorylation of titindomains, which also increases cardiac compliance and distensibility,rather than any one of the relatively unspecific vasodilatory effectsassociated with relaxin. Consequently, the method in accordance withthis aspect of the invention comprises methods for administration of thepharmaceutical composition as described in any of the claims to asubject suffering from DF, DHR or HFPEF which is the result of achemo-mechanical failure of the heart muscle.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is best understood when read in conjunction withthe accompanying figures, which serve to illustrate the preferredembodiments. It is understood, however, that the invention is notlimited to the specific embodiments disclosed in the figures.

FIG. 1 are representative diagrams showing the pressure-volumerelationship (ESPVR—end systolic pressure-volume relationship; EDPVR—enddiastolic pressure-volume relationship) and stroke volumes (SV) in caseof a systolic dysfunction (left) and a diastolic dysfunction (right);

FIG. 2 is a diagnostic flow chart in accordance with Paulus W J et al.,How to diagnose diastolic heart failure: a consensus statement by theHeart Failure and Echocardiography Associations of the European Societyof Cardiology. European Heart Journal (2007) 28(20): 2539-2550;

FIG. 3A is a plot of a completion binding experiment showing thebioequivalency of europium-labeled human relaxin-2 (cf: Shabanpoor F etal., Biochem Biophys Res. Commun. (2012) 420(2): 253-6) using humanembryonic kidney (HEK)-293T cells stably transfected with RXFP1receptor;

FIG. 3B is a plot showing the relationship between cAMP activity (cAMPreporter gene assay) in cells stably transfected with RXFP1 receptor andsynthetic human relaxin-2 (cf. Yan Y et al. in Biochemistry, 2008,47(26): 6953-6968);

FIG. 4 is a bar graph showing the endogenous venous relaxin-2concentration in patients afflicted of DHF and controls (P<0.05 versuscontrols; Mann-Whitney U test on ranks);

FIG. 5 is a bar graph showing the coronary concentration gradient ofendogenous relaxin-2 in patients afflicted of DHF and controls (analyzedwith a two-factorial ANOVA (factors: group, repeated measures; P>0.05,no significant difference);

FIG. 6 is a plot showing the coronary concentration gradient ofendogenous relaxin-2 relative to the diastolic dysfunction in patientsas determined by the echocardiographic marker E/E′ wherein E denotesearly mitral valve flow velocity and E′ denotes early myocardialrelaxation velocity as measured by tissue Doppler;

FIG. 7 is a plot showing the correlation and linear regression betweenthe left-ventricular end-diastolic pressure LVEDP (mmHg) and acirculation marker for oxidative stress, plasma nitrotyrosine (nmol/L),in two independent animal models of DHF (SH and ZDF rats—SH:spontaneously hypertensive; ZDF: Zucker Diabetic Fatty); and appropriatecontrols (Lean: Zucker lean) and controls;

FIG. 8 is a bar chart showing the decrease of the left ventricularend-diastolic pressure by an administration of synthetic relaxin-2 (5nmol/L) for 30 minutes in the animal models of FIG. 7 (Plac: placebo;Rlx: relaxin-2; SHR: spontaneously hypertensive rat; ZDF: zuckerdiabetic fatty rat; Lean: zucker lean rat; Control: control rat forSHR);

FIG. 9 is a bar chart showing the shift in titin phosphorylation fromN2B to N2BA isoform (expressed as phosphorylated titin P-N2BA/P-N2Bisoform ratio) by synthetic relaxin-2 (5 nmol/L) for 30 minutes in theanimal models of FIG. 7 (Plac: placebo; Rlx: relaxin-2; SHR:spontaneously hypertensive rat; ZDF: zucker diabetic fatty rat; Lean:zucker lean rat; Control: control rat for SHR).

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to the treatment of patients afflicted ofheart failure with preserved ejection fraction (HFPEF) and diastolicheart failure (DHF). More precisely, a relevant subgroup of thesepatients suffers from a phosphorylation deficit of the myocardialprotein titin, also known as connectin, which then leads on to a heartfailure with preserved ejection fraction (HFPEF). Titin is a largestructural protein, one of the largest known, and encoded in humans bythe TTN gene (Labeit S et al., A regular pattern of two types of100-residue motif in the sequence of titin. Nature (1990) 345: 273-6.doi:10.1038/345273a0. PMID 2129545.) With a length of about 27,000 to33,000 amino acids (depending on the splice isoform), the titincomprises roughly 200 to 250 individually folded protein domains whichunfold when the giant protein is stretched and refold when the tensionis removed. Variations in the sequence of titin between different typesof muscle (e.g., cardiac or skeletal) have been correlated withdifferences in the mechanical properties of these muscles. Thisdistinguishes the present invention from a use of relaxin or serelaxin(RLX030) aiming at a hypertension therapy and vasodilation as describede.g. by Vitovec J et al in Kardiol Rev 2013; 15(2):113-117. Thosetherapeutic application have been designed for treatment of varioustypes of acute heart failure as described herein in the backgroundsection of the invention.

With respect to the striated heart muscle, titin can be regarded workinglike a molecular spring which is not only important in the contractionof the striated muscle tissues, but also considered responsible for thepassive elasticity of muscle. There is a cardiac specific isoform oftitin. More precisely, myofilament stiffness is dependent on theexpression levels of the cardiac titin isoforms, N2B and N2BA, and bythe phosphorylation of the elastic titin domains N2-B unique sequence(N2-Bus) and PEVK. Phosphorylation of N2-Bus by cAMP-dependent proteinkinase (PKA) or cGMP-dependent protein kinase (PKG) decreases titinstiffness, whereas phosphorylation of the PEVK domain by PKC increasesit. Thus, hypo-phosphorylation of the N2-Bus and hyper-phosphorylationof the PEVK domain act complementary to elevate passive tension infailing human hearts (Müller E A et al in Cardiovasc Res (2013)doi:10.1093/cvr/cvt144; published online: Jun. 13, 2013).

The above belongs to the general technical context that the cardiacmuscle cell (myocyte) contains bundles of myofibrils which in turn aremade up of myofilaments comprising myosin and actin. The myofibrilsconsist of microanatomical units, termed sarcomeres, which represent thebasic contractile units of the myocyte. The sarcomere is defined as theregion of myofilament structures between two Z-lines. The distancebetween Z-lines (i.e., sarcomere length) ranges from about 1.6 to 2.2 μmin human hearts, whereas the myocyte is about 25 μm in diameter andabout 100 μm in length. Chemical and physical interactions between theactin and myosin cause the sarcomere length to shorten, and the myocyteto contract. Thus, the contracting sliding filaments on the one handproduce muscle contraction and titin filaments are needed on the otherhand for passive-force generation and transmission of stress. Titinstiffness however varies during heart development and disease through ashift in the expression ratio of the two main titin isoforms in cardiacsarcomeres, N2BA (compliant) and N2B (stiffer). The stiffer N2B titinisoform is inter alia triggered by thyroid hormone activating thephosphatidylinositol-3-kinase pathway. Conversely, low thyroid hormone(T3) promotes the compliant N2BA-titin. In addition, titin stiffness canbe tuned down acutely by protein kinase (PK)A- or PKG-mediatedphosphorylation of a cardiac-specific 1-band titin segment, the N2-Bdomain. Accordingly, beta-adrenergic agonists, nitric oxide, ornatriuretic peptides diminish the stiffness of titin molecular springs.Thus, an elevated passive stiffness of a human heart is likely caused bya titin N2B phosphorylation deficit which may go along with a mechanicaldysfunction of the myocardium and syndromes such as HFPEF and DHF (forreview, see Kruger et al., J Mol Cell Cardiol. (2009) 46(4):490-8;Kellermayer et al, J Muscle Res & Cell Motility (2002) 23(5-6):499-511).

The present inventors have found that the titin-N2Bus domainphosphorylation deficit may be offset by the administration of relaxin.Without being bound by any theory, it is assumed that relaxin binds tothe RLXFP1 receptor which activates the NO-cGMP-protein kinase G and/orcAMP-protein kinase A pathways in myocardial cells (fibroblasts,fibromyoblasts, cardiomyocytes, endocardial cells, endothelial cells).The increased phosphorylation activity then increases the passivecompliance of the striated heart muscle which leads to a lowering of theleft ventricular end-diastolic pressure. The relaxin protein superfamilyincludes insulin, insulin-like growth factors I and II and insulin-likefactors 3, 4, 5 and 6. Three distinct forms of relaxin have beenidentified in humans whereof relaxin-2 (H2) is the major stored form andthe only form known to be secreted into the circulation. Relaxin is aheterodimeric peptide hormone of about 6 kDa wherein an A chain and a Bchain are linked via disulphide bridges. The biological role of humanrelaxin-1 (H1) is not clear to date and relaxin-3 (H3) is found in thebrain only acting as a neuropeptide (Bathgate et al., PhysiologicalReviews 2013).

The efforts to identify relaxin receptors have led to the discovery ofthe G protein-coupled receptors LGR-7 (now classified as RXFP1) as thecognate receptor for relaxin-2 and GPCR-135 (now classified as RXFP3) asthe physiological relaxin-3 receptor. Human relaxin-2 also binds andactivates the human glucocorticoid receptor (GR) (Dschietzig T et al inFASEB J 2004; 18:1536e8). Human relaxin-1 and -2 further bind to thereceptors LGR8 (now classified as RXFP2) and GPCR142 (now classified asRXFP4). However, RXFP1 is the only widely expressed receptor which bindseach of the human relaxins (H1, H2 and H3) with high affinity. Thus, itcan be assumed that all human relaxins display similar biologicalactivity with respect to their ability to stimulate cAMP activity andother RXFP1-related pathways in cells expressing the RXFP1 receptor (forreview see Bathgate R A et al., Physiol. Rev 2013).

The intrinsic physiological function of relaxin in man is still unclear.Relaxin was originally identified by its activity as a pregnancy hormone(see Hisaw F L et al, Proc. Soc. Exp. Biol. Med. 1926; 23:661-663).Since then, relaxin has been shown to act as an endocrine and paracrinefactor causing a widening of blood vessels (vasodilatation) in thekidney, mesocaecum, lung and peripheral vasculature and to increaseblood flow and perfusion in these tissues (Dschietzig & Stangl in CMLS2002; 59:1-13 (Review); Dschietzig T et al in Circ Res. 2003; 92:32-40).A number of unrelated activities have further been attributed such as aregulator of vasotonus, plasma osmolality, angiogenesis,collagen-turnover, renal and myocardial function, and central nervousprocesses (see Dschietzig T et al in Cell. Mol. Life Sci. 2003;60:688-700; Dschietzig T et al. in Pharmacol Ther 2006; 112:38e56,Nistri S et al in Cardiovasc Hematol Agents Med Chem 2007; 5:101e8).Aberrant relaxin activity and/or expression have been implicated indiverse disorders and diseases such as high blood pressure, drinking,memory-related functions and addictive behaviors by binding thereceptors in the brain and circumventricular organs as well as forcardiovascular diseases, renal diseases, fibrotic disorders (includingcardiac fibrosis and fibrosis associated with airway remodeling),neurological disorders, immune diseases and endometrial and reproductivedisorders.

Numerous clinical applications using relaxin as well as relaxin agonistsand antagonists have therefore been suggested, particularly for treatingdiseases related to vasoconstriction (EP07008840), as a co-factor orreplacement of insulin (EP1909809), to increase arterial compliance(EP05731443), for tumor suppression (EP07719531), as adjuvant in thedifferentiation of stem cells (EP04806868), for increasing fertility(EP03005488) or control of fetal growth (EP5780049, EP98932799), formodulating apoptosis, for treating neurodegenerative dysfunctions, forpromoting angiogenesis as well as even for promoting hair growth andinhibition of cutaneous aging (EP0793505).

The treatment of the stiffened heart muscle however is a medicalindication clearly independent from the regulation of the vasotonus orother renal and myocardial functions. The instant pharmaceuticalcomposition comprises relaxin molecules, more preferably humanrelaxin-2, which bind the RXFP1 receptor present on fibroblasts,fibromyoblasts, endothelial cells, endocardial cells, and cardiomyocytesin the myocardium (i.e., the entire cardiac muscle consisting ofdifferent cell types). The experiments hereinafter indicate thatpatients suffering from a titin N2B phosphorylation deficit and astiffened heart muscle can obtain thereby an increase of the heart'sstroke volume at lower pressures. Until this date, there has been notherapy of a diastolic stiffened heart muscle which leads to anincreased distensibility and compliance of the heart muscle without theundesirable circumstances of a dilated heart.

More precisely, the heart muscle in diastolic heart failure (HFPEF) mustbe distinguished from a heart muscle in systolic heart failure (HFREF,heart failure with reduced ejection fraction). A dilated heart in HFREFhas increased volume relative to the amount of diastolic pressure. Incontrast, hearts in diastolic heart failure (HFPEF) show increasedfilling pressures already at normal or even slightly reduced volume (seealso the diagrams in FIG. 1 depicting systolic [left] and diastolicheart failure [right]). Clinically, both different entities of heartfailure, HFPEF and HFREF, may exhibit similar symptoms (edema, dyspneascongestion, fatigue). The fact that in some patients suffering fromsystolic heart failure (HFREF), a diastolic dysfunction can also bedetected does not weaken the fundamental differences: In systolic heartfailure (HFREF), diastolic dysfunction might occur and is caused by andrelated to the leading systolic impairment. In diastolic heart failure(HFPEF), diastolic dysfunction in combination with a generally stiffenedvascular bed prevails by far. These pathophysiological considerationscorrespond to the clinical and therapeutical situation: Contemporarymedicine has at its disposal a remarkable variety of drugs(angiotensin-converting enzyme inhibitors, angiotensin receptorblockers, beta blockers, aldosterone antagonists, ivabradine) that lowermortality in systolic heart failure, but not one such drug exists forHFPEF despite absolutely comparable and even growing epidemiologicalrelevance (Yancy et al. in JACC 2006; Aurigemma G P et al. in NEJM(2004) 351:1097-1105). Many drugs beneficial in HFREF have alreadyfailed in large clinical studies in diastolic heart failure:angiotensin-converting enzyme inhibitors and angiotensin receptorblockers failed in the PEP-CHF Trial (using perindopril), theCHARM-preserved Trial (candesartan), and in the I-PRESERVE Trial(irbesartan); beta blockers in the ELANDD Trial (nebivolol). Trials withaldosterone antagonists (RAAM-PEF testing eplerenone, ALDO-DHFevaluating spironolactone) have produced mixed results in recentphase-II trials and are far from having shown any mortality benefit.

Heart failure with preserved ejection fraction (HFPEF) is no raredisease. Up to half of all patients presenting clinical signs of heartfailure suffer from diastolic heart failure and this particular entityis characterized by a normal ejection fraction (preserved systolicfunction) but increased left ventricular filling pressures and clinicalsigns of heart failure (Yancy C W et al. in J Am Coll Cardiol. (2006)47(1):76-84). Mortality in this entity of heart failure is comparable tothat in systolic heart failure (Owan et al. in NEJM 2006, 355:251-259)so that a therapeutic treatment using a relaxin composition will achievea favorable impact on the morbidity and mortality rates. Notably elderlypatients (>70 yrs age) and patients with hypertension or diabetes maysuffer from a titin phosphorylation deficit and a stiffened diastolicheart and they would benefit from a therapeutic treatment comprising anadministration of relaxin. Thus, the need for a relaxin treatment isrising with the expectable demographic change in our society (Smith G Let al. in J Am Coll Cardiol (2003) 41:1510-8).

The disclosed composition preferably comprises a physiologically activeamount of human relaxin molecules, most preferably a physiologicallyactive amount of human relaxin-2 (H2) or a fusion protein with theactive core of human relaxin which binds the RXFP1 receptor. U.S. Pat.Nos. 4,758,516 and 4,871,670 (Hudson et al) disclose the gene andprotein sequences of human relaxin H1 and H2. Methods of synthesizingrelaxin are described in U.S. Pat. No. 4,835,251 (Burnier et al), WO2010/140060 (Barlos) and W02013/17679 (Dschietzig T et al). Bioassayswith synthetic relaxin H2 and analogues thereof have revealed a relaxincore structure bearing the biological activity (Dschietzig et al. inPharmacol Ther. 2006; 112:38-56). A preferred embodiment thereforerelates to a pharmaceutical composition comprising as active ingredienta compound having a relaxin core that binds to the RXFP1 receptor.

Recombinant H2 have already been tested in acute cardiovascular therapyand for treating neurodegenerative diseases (see U.S. Pat. No. 5,166,191(Cronin et al) and Dschietzig T et al in. Journal of Cardiac Failure(2009) 15:182-190; Teerlink et al. in Lancet 2012, pii:S0140-6736(12)61855-8; doi: 10.1016/S0140-6736(12)61855-8). Thecardiovascular activity of relaxin-2 has further been evaluated inanimal models (Perna A M et al. (2005) in FASE J, 19: 1525-1527; Bani,D. et al. in Am. J. Pathol. (1998) 152:1367-1376; Zhang J. et al. inPeptides (2005) 26: 1632-1639). These studies examine cardiacdysfunctions such as cardiomyopathy, diabetic cardiomyopathy, myocardialinfarction or isoprenaline-induced cardiac toxicity for whichestablished therapies are available, for example, an administration ofangiotensin-converting enzyme inhibitors, angiotensin II receptorblockers, calcium channel blockers, diuretics, vasodilators, and betablockers. A treatment of a stiffened diastolic heart muscle does notbelong to these therapies. There have been also no successful clinicaldata concerning the treatment of HFPEF, cardiac fibrosis, valvulardysfunctions (abnormal thickening of the heart valves) and/or a fibroticcardiac muscle may be concomitant, but unrelated medical indications.The latter is caused by over-activated fibroblasts and fibromyoblasts,not normally but excessively secreting collagen which leads to a loss offlexibility and distensibility of the heart muscle. A reduced structuralsupport may however lead to a “dilated” or “remodeled” heart as alsoobserved for drugs binding to the serotonin (5-hydroxytryptamin)receptor. While the administration of relaxin-2 reduces excessivecollagen secretion and accumulation, it is pointed out that theactivation of RXFP1 receptor on fibroblast and fibromyoblasts does notaffect the basal collagen content in healthy tissues so that theadministration of a RXFP1 agonist such as human relaxin-2 does not giverise to a dilated heart but is safe for therapeutic use in man.

Human relaxin-2 has also been tested as a peripheral and renalvasodilator, for increasing glomerular filtration rate and renal bloodflow, and for augmenting endothelin type B receptor-mediatedvasodilation and endothelin-1 clearance while it also stimulates therelease of atrial natriuretic peptide. Others have shown that theadministration of porcine relaxin induces favorable hemodynamic changesin intact rats, decreasing afterload and elevating the cardiac indexwithout a significant drop of arterial pressure. Moreover, relaxin hasbeen tested in human clinical trials as a therapeutic option forscleroderma patients and patients having chronic systolic heart failure(Seibold et al. in Annals of Internal Medicine 2000, 132:871-879;Dschietzig et al. in J of Cardiac Failure 2009, 15:182-190. Thevasodilatory effects of relaxin have further been examined in studieswith patients having acute decompensated heart failure (ADHF) andchronic systolic heart failure (CHF). These are again other medicalindications for which other therapies are available and do not relate tothe medical subgroup of chronic HFPEF due to altered chemo-mechanicalcharacteristics of the proteins (titin) in the myocardium followingoxidative stress or aging.

The present disclosure concerns the use of relaxin-2 for treatingpatients affected by a heart failure with preserved ejection fraction(HFPEF) and not in need of the vasodilatory activities of relaxin. Thisdistinguishes the present invention from disclosures based on thevasodilatory effects of relaxin (WO 2013/017679). The latter activitiesresult from a vascular activity occurring on endothelial cells and cellsof the vascular smooth muscles such as found in the tunica media layerof large (aorta) and small arteries, arterioles, vein, as well as inlymphatic vessels, the urinary bladder, uterus (termed uterine smoothmuscle), male and female reproductive tracts, gastrointestinal, tract,respiratory tract and around the glomeruli of the kidneys (thesespecialized smooth muscle-like cells are called mesangial cells).

While smooth muscle cells have basically the same structure and functionin different organs, the cardiac muscle is a type of striated muscle anddifferent to the skeletal and other smooth muscles. Thus, the presentdisclosure relates to an activity of human relaxin-2 mediated by therelaxin receptor (RXFP1) present on fibroblasts, fibromyoblasts,endothelial cells, endocardial cells, and cardiomyocytes in themyocardium. Such has not been suggested or described previously. In linewith this, the N2B isoform of titin which is the therapeutic target ofhuman relaxin-2 in the present disclosure is absolutely cardio-specificand not found in any other types of muscles (see for review Aronson andKrum, Pharmacol Therap 2012).

Chagas' heart disease is further said to represent a model of diastolicheart failure that spares systolic function (Marin-Neto et al. inEvidence-Based Cardiology, 3rd Ed., Yusuf S, Cairns J, Camm J et al(EDS), 2010, p823ff). It is therefore contemplated using a relaxin-basedcomposition as described for a treatment of Chagas myocarditis, say ofpatients seropositive for Trypanosoma cruzi in order to preserve thedistensibility of the heart muscle and to counter formation of a dilatedheart. Without being bound by any theory it is contemplated that theTrypanosoma parasite causes a phosphorylation deficit and interfereswith the structural properties of the titin isoforms.

Diagnosis of Diastolic Heart Failure (2013ICD-10-CM 150.3+4)

A patient is principally diagnosed having a diastolic dysfunction(HFPEF) when having the signs and symptoms of heart failure but ameasured left ventricular ejection fraction close to normal or above60%. Another diagnostic tool is an elevated BNP level in combinationwith a normal ejection fraction. Echocardiography may be used todiagnose diastolic dysfunction but no one single echocardiographicparameter can confirm a respective diagnosis. Multiple echocardiographicparameters have been proposed as sensitive and specific, includingmitral inflow velocity patterns, pulmonary vein flow patterns, E:Areversal, tissue Doppler measurements (i.e., E/E′ ratio), and M-modeecho measurements (i.e. of left atrial size). Algorithms have furtherbeen developed which combine multiple echocardiographic parameters.

There are four basic echocardiographic patterns of diastolicdysfunction, which are graded I to IV: The mildest form is called an“abnormal relaxation pattern”, or grade I diastolic dysfunction. On themitral inflow Doppler echocardiogram, there is reversal of the normalE/A ratio. This pattern may develop normally with age in some patients,and many grade 1 patients will not have any clinical signs or symptomsof heart failure. Grade II diastolic dysfunction is called “pseudonormal filling dynamics”. This is considered moderate diastolicdysfunction and is associated with elevated left atrial fillingpressures. These patients more commonly have symptoms of heart failure,and many have left atrial enlargement due to the elevated pressures inthe left heart. Grade III and IV diastolic dysfunction are called“restrictive filling dynamics”. These are both severe forms of diastolicdysfunction, and patients tend to have advanced heart failure symptoms:Class III diastolic dysfunction patients will demonstrate reversal oftheir diastolic abnormalities on echocardiogram when they perform theValsalva maneuver. This is referred to as “reversible restrictivediastolic dysfunction”. Class IV diastolic dysfunction patients will notdemonstrate reversibility of their echocardiogram abnormalities, and aretherefore said to suffer from “fixed restrictive diastolic dysfunction”.The presence of either class III or IV diastolic dysfunction isassociated with a significantly bad prognosis. These patients will haveleft atrial enlargement. Imaged volumetric definition of systolic heartperformance is commonly accepted as ejection fraction. Volumetricdefinition of the heart in systole was first described by Adolph Fick ascardiac output. Fick may be readily and inexpensively inverted tocardiac input and injection fraction to mathematically describe adiastolic dysfunction. Decline of injection fraction paired with declineof the E/A ratio and an increase of the E/E′ ratio seems a strongerargument in support of a mathematical definition of diastolic heartfailure. Regardless of any systolic dysfunction and severity, adiastolic dysfunction can be beneficially treated using a relaxin-basedcomposition when the decreased compliance and distensibility of theheart muscle is caused by a phosphorylation deficit of the relevanttitin isoforms.

Diagnosis of an Altered Phosphorylation Pattern of Titin (Titin N2BIsoform).

The titin exon 49 isoform (Genbank AJ277892—Freiburg A et al.,Circulation Research, 86, 1114-1121) is for example released into thecirculation in case of a damaged heart. The amino acid sequence of titinexon 49 is cardiospecific since no such isoform occurs in other skeletalor smooth muscles. The cardiac specific titin exon 49 sequence comprises928 amino acids (exon 49) and can be detected in serum and isolatedtherefrom. The detection limit for serum titin N2B protein is about 10μg/mL and there are monoclonal antibodies available which can be used ascapture antibodies for immunological purification of titin N2B fromserum (DE 10 2012 017 566.3—Labeit D et al.). The purified titin N2B maythen be examined for its phosphorylation using electrophoresis (BorbelyA et al. in Circ. Res 2009, 104(6): 780-6) or preferably massspectrometry (Kötter S et al in Cardiovasc Res 2013, doi:10.1093/cvr/cvt/44).

Other indirect markers for a titin phosphorylation deficit may beincreased levels of plasma or serum nitrotyrosine and interleukin-6,both markers for oxidative stress. Patients with diastolic heart failurealso have significantly increased plasma levels of tumor necrosis factoralpha and interleukin 6 and 8 compared with control subjects.Nitrotyrosine expression, a measure of nitrosative/oxidative stress,correlates in particular with myocardial protein kinase G activity (PKG)and downstream with the titin phosphorylation activity incardiomyocytes. It has already previously been speculated that thecorrection of the myocardial protein kinase G activity could be a targetfor specific HFPEF treatment but no pharmaceutical composition to thiseffect had been proposed. In this context, human relaxin-2 is uniquebecause it directly stimulates the cGMP-PKG path by increasing nitricoxide, but also indirectly protects the bioavailability of nitric oxideand the functional integrity of soluble guanylate cyclase (i.e., theenzyme producing cGMP upon stimulation by nitric oxide) by itsanti-oxidative and anti-inflammatory effects (Dschietzig T et al.,Cardiovasc Res 2012).

EXAMPLES Example 1—Synthetic Human Relaxin-2 and Bioequivalency (PriorArt)

The human relaxin-2 was prepared as disclosed in WO 2013/17679(Dschietzig T et al) using a modification of the Merrifield method(Merrifield R B. Solid phase synthesis (Nobel Lecture) Angew. Chem Int.Ed. 1985; 24: 799-810). In essence, the process for preparing humanrelaxin-2 comprised the solid state synthesis of the following aminoacid sequences:

-A chain SEQ ID NO: 1 pGlu-Leu-Tyr-Ser-Ala-Leu-Ala-Asn-Lys-Cys-Cys-His-Val-Gly-Cys-Thr-Lys-Arg-Ser-Leu-Ala-Arg-Phe-Cys -B chain: SEQ ID NO: 2Asp-Ser-Trp-Met-Glu-Giu-Val-Ile-Lys-Leu-Cys-Gly-Arg-Glu-Leu-Val-Arg-Ala-Gln-Ile-Ala-Ile-Cys-Gly- Met-Ser-Thr-Trp-Ser

For an intrachain bridge of the A chain (Cys-10 with Cys-15) and thecombination the A and B chain (Cys-11 of A with Cys 11 of B and Cys-24of A with Cys-23 of B) the A and B chains were first synthesized usingtrityl-protected cysteines (L-Cys(Trt)-OH). The individual chains A andB were purified after solid state synthesis by chromatography followedby simultaneous folding and combination of the individual chains A and Bin an ammonium hydrogen carbonate buffer at pH 7.9 to 8.4; andsubsequent purification of the relaxin-2 formed.

The prepared peptide hormone was structurally identical andbioequivalent to human relaxin-2 and recombinant human relaxin-2.Experiments were conducted in HEK293T cells over-expressing the RXFP1receptor, an established cell line for basic receptor pharmacology, andin THP-1 cells, a human macrophage cell line with endogenous RXFP1expression. The binding properties were determined using respectivecells plated on 96-well IsoPlate™ microplates (PerkinElmer) with whitewalls and clear bottoms precoated with poly-L-lysine. The comparativebinding was determined with europium-labeled human relaxin-2 as tracerand increasing concentrations of synthetic human relaxin-2 (twodifferent lots). The unspecific binding was determined in the presenceof an excess (500 nM) of unlabeled human relaxin-2. Each concentrationpoint was determined in triplicate. The peptides were tested in at leastthree independent assays to confirm activity. Curves were fitted using aone site binding model in GraphPad Prism 4.0 (GraphPad Software, SanDiego, Calif., USA). The inhibition constants (KI) as a measure ofpeptide activity were determined from the IC₅₀ values using theCheng-Prusoff equation.

HEK-293T cells stably expressing RXFP1 and a pCRE-11-galactosidasereporter plasmid were further employed to determine the ability ofsynthetic human relaxin-2 (shRlx) to activate RXFP1-related signaling(Halls M L et al. in Ann NY Acad Sci. 2009; 1160:108-11). Stimulation ofthe RXFP1 receptor results in the activation of adenylate cyclase andtherefore in an increase in cAMP. Cells were incubated for 6 hours withincreasing concentrations of synthetic human relaxin-2 of two differentlots and relaxins from other sources as indicated. Each concentrationpoint was performed in triplicate and peptides were tested in at leastthree independent experiments. Data were analyzed with GraphPad Prism4.0 (GraphPad Software, San Diego, Calif., USA), and a nonlinearregression sigmoidal dose-response (variable slope) model was used toplot curves and calculate pEC₅₀ values.

The synthetic human relaxin 2 peptide bound equally as recombinant humanrelaxin-2 to human relaxin receptor RXFP1 (relaxin family peptidereceptor 1); see Table 1 and FIG. 3A. Signal transduction was alsosimilar as shown by the cAMP assays developed for human relaxin-2; seeFIG. 3B. On human THP-1 cells, synthetic and recombinant human relaxin-2showed equivalent bioactivities; see Table 1 below for details:

TABLE 1 Testing of different lots of hRLX-2 for RXFP1 binding and cAMPgeneration RXFP1 binding cAMP response Peptide pKi pIC50 pEC50 HEK293Tcells overexpressing RXFP1 shRlx, lot 01 8.9 ± 0.12 (3) 8.8 ± 0.11 (3)10.1 ± 0.12 (3) shRlx, lot 02 9.3 ± 0.15 (3) 9.2 ± 0.15 (3) 10.4 ± 0.04(3) -Asp-shRlx 10.2 ± 0.03 (4) rhRlx, Bathgate 2006 10.2 ± 0.26 (3) 10.6 ± 0.04 (4) rhRlx, Hossain 2008 9.2 ± 0.16 (3) 10.4 ± 0.04 (4)rhRlx, Yan 2008  9.4 ± 0.11 (10)  10.6 ± 0.06 (16) rhRlx, Halls 2009 9.6± 0.17 (4)  9.4 ± 0.22 (7) rhRlx, Hossain 2011 9.0 ± 0.06 (8) 10.3 ±0.04 (5) rhRlx, Scott 2012 8.8 ± 0.05 (3) 10.7 ± 0.04 (3) rhRlx, Chan2012 9.7 ± 0.07 (5) 10.7 ± 0.16 (5) THP-1 cells shRlx, lot 01  9.1 ±0.19 (3) shRlx, lot 02  9.2 ± 0.32 (3) rhRlx, Halls 2009  9.3 ± 0.12 (6)Table 1: Mean ± standard deviation (number of experiments inparentheses) of pKi and pIC50 values reflecting RXFP1 binding and ofpEC50 reflecting cAMP generation. pKi: negative decadic logarithm of theInhibition constant Ki; pIC50: negative decadic logarithm of the 50%inhibitory concentration; pEC50: negative decadic logarithm of thehalf-maximum concentration; shRlx: synthetic human relaxin-2 of example1; -Asp-shRlx is the single aa degradation product of synthetic humanrelaxin-2; rhRlx: human relaxin-2 prepared by recombinant cells.

Moreover, in an established cell model of myocardial hypertrophy(Dschietzig T et al. in Pharmacol. Ther. 2006; 112: 38-56) synthetichuman relaxin-2 proved as potent as recombinant human relaxin-2. In ourmodel, synthetic human relaxin-2 inhibited the differentiation ofcardiac fibroblasts into myofibroblasts and the secretion of growthfactors by these cells. The synthetic human relaxin-2 had excellentstability. A biologically fully active by-product without the N-terminalaspartate of the B chain was observed after 80 days at 37 degreesCelsius.

Example 2—Safety and Dose Response (First Medical Indication—Prior Art)

Human relaxin is physiologically up-regulated and plays a compensatoryrole in human heart failure like B-type natriuretic peptide. Fordetermining the safety and dose response to human relaxin (recombinantlyproduced human relaxin-2, rhRLX) in stable patients with heart failuresixteen patients were treated with intravenous human relaxin-2 in threesequential dose cohorts and monitored hemodynamically during the 24-hinfusion and post-infusion periods.

The safety demonstrated in group A (treatment for 8 h each with dosagesequivalent to 10, 30, and 100 μg human relaxin-2/kg/day) allowedescalation to group B (240, 480, and 960 μg/kg/day), and the highestsafe dose, 960 μg/kg/day, was selected for a 24-h dosing in group C.

The human relaxin-2 showed no relevant adverse effects and producedhemodynamic effects consistent with systemic vasodilation, i.e., trendstoward increases in the cardiac index and decreases in pulmonary wedgepressure, without inducing hypotension. Thus, the demonstratedhemodynamic effects of human relaxin-2 represent a first therapeutic useof intravenously administered human relaxin-2 in human heart failure,notably congestive heart failure (Dschietzig et al. in FASEB Journal2001, 15:2187-2195; BfArM approved study, A Pilot Safety andDose-Finding Trial of Intravenous Recombinant Human Relaxin (rhRlx) inCompensated Congestive Heart Failure”, EudractCT 2005-001674-27,Protocol No RLX-CHF.001). An intravenous administration of humanrelaxin-2 has also been favorably tested in clinical phase III for itshemodynamic effects in cases of acute heart failure (Teerlink et al.,Relaxin for the treatment of patients with acute heart failure(Pre-RELAX-AHF): a multi-centre, randomised, placebo-controlled,parallel-group, dose-finding phase IIb study. Lancet 2009,373:1429-1439; (6) Ponikowski et al., Design of the Relaxin in acuteheart failure study. American Heart Journal 2012, 163:149-155).Consequently, there are sufficient data that human relaxin-2 can besafely administered for treatment of heart failures.

Example 3—Treatment of Diastolic Dysfunction by Subcutaneous Infusion ofRelaxin

Relaxin is known playing a role in the transformation of fibrocytes tofibromyoblasts, the initial step for the development of tissue fibrosis,and to modulate and attenuate the stimulated secretion of matrixproteins by fibromyoblasts (Samuel et al. in Endocrinology 2004,145:4125-4133). Thus, relaxin may also be used to mitigate myocardialhypertrophy (Dschietzig et al in Annals of the NY Acad, of Science 2005,1041:441-3; Moore et al. in Endocrinol. 2007, 148:1582-1589; Samuel etal., in Endocrinology 2008, 149:3286-3293; Lekgabe et al. inHypertension 2005, 46:412-418; Xioa-Jun Du et al in Cardiovascular Res2003, 57:395-404). These disclosures however relate to processes due toa fibrotic or hypertrophic stiffening of the heart muscle, unrelated tothe chemo-mechanical stiffening of the muscle proteins.

The present inventors have therefore used an animal model for testingthe treatment for DHF and of a stiffened heart muscle havinginsufficient relaxation and distensibility. The table below summarizesdata obtained in mice with a type of diastolic heart failure as comparedwith controls. The subcutaneous infusion of isolated porcine relaxinover 12 weeks in mice (50 μg porcine relaxin/kg/d) decreasedend-diastolic pressure and, consequently, improved E/A ratio, afunctional parameter indicating the quality of ventricular filling. Thesubcutaneous infusion of relaxin was obtained by implanting in the neckportion of the mice a membrane capsule releasing continuously porcinerelaxin. The results have been summarized in Table 2 below:

TABLE 2 Treatment of diastolic dysfunction by subcutaneous infusion ofrelaxin Controls Diastolic Dysfunction Baseline Baseline BaselineBaseline before Placebo before Relaxin before Placebo before RelaxinPlacebo 12 weeks Relaxin 12 weeks Placebo 12 weeks Relaxin 12 weeks (n =15) (n = 15) (n = 15) (n = 15) (n = 15) (n = 15) (n = 15) (n = 15) LVSPNd 121 ± 13  Nd 109 ± 10  Nd 112 ± 13  nd 111 ± 12  LVEDP Nd 5.0 ± 0.4Nd 5.2 ± 0.5 Nd 10.7 ± 1.0* Nd  7.0 ± 0.6*§ E/A 2.5 ± 0.2 2.3 ± 0.3 2.6± 0.2 2.4 ± 0.2 1.5 ± 0.1*  1.4 ± 0.2* 1.6 ± 0.1* 2.0 ± 0.2# ratio Data,given as mean ± SEM, were obtained in mice. LVSP (mm Hg): leftventricular systolic pressure as measured by invasive micromanometry;LVEDP (mm Hg): left ventricular end-diastolic pressure as measured byinvasive micromanometry; E/A ratio: ratio of echocardiographicallydetermined mitral flow velocities. *P < 0.05 compared with controls; #P< 0.05 compared with baseline (Friedman ANOVA on ranks); §P < 0.05compared with placebo; nd: not determined - as the mice had to besacrificed after catheterization, no baseline catheter measurementscould be made prior to administration of placebo or relaxin.

The data show that the subcutaneous infusion of porcine relaxin had abeneficial effect on the heart function of diastolic mice only whereasno pharmaceutical effects were noted in the controls. Thus, relaxinproved effective when the performance of the heart was alreadyendangered by stiffening actions. In the controls the measured E/A ratiowas not affected in any way (E/A=2.5 or 2.6±0.2). However, in case of adiastolic dysfunction the reduced E/A ratio dramatically shifted back tothe normal range showing that the administration of relaxin has greatlyimproved the diastolic functions of the mice hearts. Thus, the datasupport that the treated hearts had improved distensibility andrelaxation. Notwithstanding, there was a need to understand the effectsof relaxin and the underlying mechanism to this observation.

Example 4—Myocardial Production of Endogenous Human Relaxin-2

Among patients undergoing elective catheterization for ablation ofatrial fibrillation, we recruited 20 control patients (5 postmenopausalwomen, 15 men) without systolic or diastolic heart failure and 30patients (10 postmenopausal women, 20 men) suffering from diastolicheart failure (DHF) as diagnosed using the diagnostic algorithmaccording to Paulus et al., see FIG. 2.

Blood for determination of relaxin-2 by ELISA was drawn immediatelybefore the ablation procedure from antecubital vein, aorta/leftventricle, and coronary sinus. The concentration difference betweenaorta/left ventricle and coronary sinus was termed the coronarygradient. A positive gradient (coronary sinus level>aortic/leftventricular level) was considered a measure of myocardial relaxinproduction, a negative one was indicative of myocardial consumption ofcirculating relaxin.

Circulating (venous) relaxin-2 was significantly increased in patientswith diastolic heart failure (DHF) as compared with controls; see FIG. 4(P<0.05 versus controls; Mann-Whitney U test on ranks). Regarding thecoronary gradient of relaxin-2 we did not find any significantdifference between controls and DHF patients; see FIG. 5 (Analyzed witha two-factorial ANOVA (factors: group, repeated measures); P>0.05, nosignificant difference). However, when we analyzed the relation betweencoronary relaxin-2 gradients and an echocardiographic measure ofdiastolic dysfunction (E/E′, see below) in DHF patients, we found ahighly significant non-linear inverse correlation. In other words, thehearts of DHF patients with high relaxin gradients (reflecting highmyocardial production) perform better in diastole than those of DHFpatients with low or even negative gradients. The results of thisanalysis are shown in FIG. 6: Individual coronary gradients of relaxin-2in DHF patients in dependence of diastolic function as assessed by E/E′.E/E′ is the most established echocardiographic marker of diastolicdysfunction which is used to classify and diagnose DHF (see Paulus etal., Eur Heart J 2007; FIG. 2). E denotes early mitral valve flowvelocity, E′ denotes early myocardial relaxation velocity as measured bytissue Doppler; E/E′ is the dimensionless ratio thereof. Cubicregression with 95% confidence intervals; Pearson correlationcoefficient r=0.95. In controls, no such correlation was found.

These findings give rise to the conclusion that the individual lack ofmyocardial relaxin-2 up-regulation worsens the individual degree ofdiastolic dysfunction. Vice versa, therapeutic relaxin administration isexpected to improve DHF significantly. Most importantly, it should benoted that human relaxin-2 affects the fibroblasts and myofibroblast ofthe heart and that these beneficial effects have no hemodynamic origin,say do not come from the vasodilatory effects of human relaxin-2.

Example 5—Correlation Between Inflammation/Oxidative Stress and LeftVentricular End Diastolic Pressure and Altered Titin N2BHypophosphorylation by the Administration of Relaxin-2

Hearts from male 5-month old control and spontaneously hypertensive(SHR) rats as well as from 12-week old Zucker Lean (Lean) and ZuckerDiabetic Rats (ZDF) were excised and mounted as Langendorff preparations(n=16 each). Similarly, hearts were also taken from wild-type (WT) andheterozygous 112-adrenoreceptor transgenic (TG) mice generated fromheterozygous (C57Blk6JxSJL) parents (n=10 each). The 112-adrenoreceptortransgenic (TG) mouse is a model with prevailingly fibrosis-relateddiastolic dysfunction.

During prior exsanguination of the animals, blood had been taken for thedetermination of plasma nitrotyrosine and interleukin-6 (IL-6). The leftventricular end-diastolic pressure (LVEDP) at maximum left ventricularsystolic pressure development (active LVP) was determined. Thereafter,all hearts were treated with 5 nmol/L synthetic human relaxin-2 orplacebo for 30 min (n=8 for each subgroup). Eventually, the total andphosphorylated N2BA and N2B titin isoforms were detected and quantifiedin homogenates of the free left ventricular wall.

Baseline:

At maximum active LVP, SHR hearts showed significantly elevated LVEDP ascompared with control hearts: SHR, 14±2 mm Hg; control, 5±1 mm Hg.Likewise, ZDF hearts had higher LVEDP than Lean hearts: ZDF, 12±2 mm Hg;Lean, 6±1 mm Hg. P<0.01; and TG mice had significantly higher LVEDP thanWT mice: TG, 12.3±2 mm Hg; WT, 4.9±1.1 mm Hg (Kruskal-Wallis ANOVA onranks followed by Mann-Whitney U-test).

Plasma levels of nitrotyrosine and IL-6 correlated with baseline LVEDP,both in the control-SHR and in the Lean-ZDF cohorts of rats. FIG. 7shows this correlation in the rat groups for nitrotyrosine and LVEDP;Pearson's correlation coefficient was r=0.89 over all rat groups(P<0.01). Likewise, a positive correlation between plasma IL-6 and LVEDPwas found, with r=0.91 over all rat groups (P<0.01). No such correlationwas found in the WT and TG mice.

Effect of Relaxin-2 on LVEDP (Treatment)

As compared with placebo, 5 nmol/L of synthetic human relaxin-2administered for 30 min markedly decreased LVEDP in the SHR and ZDFhearts whereas smaller changes were seen in control and Lean hearts. InWT and TG mice, synthetic human relaxin-2 evoked small, but significantLEVEDP decreases which, in contrast to the data obtained in the two ratmodels, did not differ between controls (WT) and diseased animals (TG).The bar chart of FIG. 8 summarizes the resulting physiological effect onthe heart in terms of the mean decreases in LVEDP in the differentgroups (* indicates P<0.05 vs placebo; **, P<0.01, Kruskal-Wallis ANOVAon ranks followed by Mann-Whitney U-test). The administration ofrelaxin-2 had clearly a substantive beneficial effect on hearts of ZDFand SH rats which suffered from oxidative stress and altered mechanicalproperties of the cardiac muscle.

Effect of Relaxin on Titin Phosphorylation

In contrast to placebo, 5 nmol/L of synthetic human relaxin-2administered for 30 min significantly reversed the relativehypophosphorylation of the stiff titin N2B isoform (which is expressedhere as ratio P-N2BA/P-N2B) found in SHR and ZDF hearts. In contrast,the relative phosphorylation status of N2B titin was not changed in TGas compared with WT mice (comparison of the WT and TG placebo groups),and the relaxin effect did not differ between controls (WT) and diseasedanimals (TG). FIG. 9 summarizes the mean P-N2BA/P-N2B ratios in thedifferent groups (* indicates P<0.05 vs placebo; **, P<0.01,Kruskal-Wallis ANOVA on ranks followed by Mann-Whitney U-test).

In all rat groups examined, but not in mice, we found a significantcorrelation between the P-N2BA/P-N2B ratio of the placebo-treated heartsof all groups and baseline LVEDP (Pearson's r=0.75 over all groups[P<0.01]) Thus, the increased baseline LVEDP found in hearts with DHFcorrelates with the relative N2B phosphorylation deficit (P-N2BA/P-N2Bratio) and the baseline LVEDP can be lowered by the administration ofrelaxin-2 which decreases the P-N2BA/P-N2B ratio by increasing the N2Bphosphorylation.

Discussion of Results

The giant protein titin poses the major determinant of myocardialdiastolic stiffness (Borbely et al., Circ Res 2009; 104:780-786; Paulusand Tschoepe, JACC 2013; 62:263-271). An increased myocardial diastolicstiffness with concomitantly increased left ventricular filling pressure(LVEDP) is further a most clear diagnostic sign for the presence ofHFPEF (diastolic heart failure, diastolic dysfunction) and that HFPEF(heart failure with preserved ejection fraction, HFPEF) can not only berelated to a hypophosphorylation of the stiff N2B titin isoform but thatan impaired titin phosphorylation is attributable to increased oxidativestress which compromises the bioavailability of nitric oxide. It haspreviously been shown that the latter diminishes cGMP-protein kinase Gsignaling and N2B phosphorylation (Heerebeek et al., Circulation 2012;126:830-839). The present inventors have further presented evidence thatsynthetic human relaxin-2 is capable of increasing the cAMP activity inRXFP1 cells, which can offset a phosphorylation deficit of the titinisoforms, which also leads to an improvement (lowering) ofpathologically elevated LVEDP in two independent animal models ofdiastolic dysfunction, SHR and ZDF. It was further demonstrated for thefirst time that the decreasing effect of relaxin on LVEDP is accompaniedby elevated N2B phosphorylation. In our models, the circulating markersof oxidative stress and/or inflammation, nitrotyrosine andinterleukin-6, correlated with the degree of diastolic dysfunction(LVEDP increase) and with the degree of relative N2Bhypophosphorylation. On the other hand, in the model with prevailinglyfibrosis-related diastolic dysfunction, the ß2-adrenoreceptor transgenicmouse, there is not phosphorylation deficit of N2B titin which could betreated by the administration of relaxin. Thus, the effects of syntheticrelaxin-2 did not differ between controls and diseased animals.Accordingly, synthetic human relaxin-2 is a candidate drug to treatpatients suffering from DHF who demonstrate high plasma levels ofoxidative/inflammatory markers indicative of increased diastolicmyocardial stiffness owing to titin N2B hypophosphorylation.

Example 7—Testing of DHF Patients (Clinical Phase II) with SyntheticHuman Relaxin-2

The official approval of the clinical phase II is being obtained forpatients diagnosed of diastolic heart failure as described by Paulus etal., see diagram of FIG. 2. The criteria for inclusion in the DHF studyare a) hospitalization for dyspnea within the last six months; b) astable medication for at least four weeks prior treatment; c) a leftventricular ejection fraction (LVEF)—preserved ejection fraction of atleast 50%; d) an established diagnosis of HFPEF according to Paulus etal. (see diagram with diagnostic flow chart, FIG. 2). Excluded from thestudy are patients having any relevant systolic dysfunction, or an acutecoronary syndrome within the last 4 weeks, or an acute myocardialInfarction or a heart surgery within the last six months, or hypotension(a systolic blood pressure of less than 100 Hg), or life-threateningtachycardia or bradycardia within the last six month prior study; or aserum creatinine of more than 2 mg/dl prior study; pulmonary disordersor dyspnea of non-cardiac origin; or any malignancies.

Three groups of twelve patients receive a) a placebo, b) a low dose ofhuman relaxin-2 at 10 μg/kg/d or c) high doses of relaxin at 100 μg/kg/dfor a period of three months, including 4 weeks of a follow-upmonitoring. The study is being done at one hospital, double-blind,prospective and randomized.

The following additional evaluations are being done or have beencontemplated:

-   -   Echocardiographic evaluation of the diastolic function (E/E′,        LAVI) following relaxin administration    -   Spiroergometry for assessing exercise capacity and associated        restrictive factors    -   Measurement of serum NT-proBNP as “companion marker”    -   Measurement of circulating IL-6, IL-8, nitrotyrosine as        inflammation and companion markers    -   Determination of collagen turnover (PINP, PIIINP, MMP-2, TIMP-4,        PIIINP, MM P-8)    -   QOL and Dyspnea questionnaires    -   Evaluation of different modes of relaxin administration: s.c.        oder i.v. (PLGA-Galenik).    -   Monitoring “Safety/Efficacy”: day 7, 30, 60, 90 and 120

1. A method for increasing cardiac inflow by administering to a patientexhibiting pathologically diminished cardiac inflow a therapeuticallyeffective amount of a compound capable of specific binding to therelaxin receptor (RXFP1) in the myocardium and lowering the heart'send-diastolic pressures and increasing the heart's stroke volume.
 2. Themethod of claim 1, wherein the compound capable of specific binding tothe relaxin receptor (RXFP1) comprises any one of the peptide sequencesSEQ ID NO: 01 and SEQ ID NO:
 02. 3. The method of claim 1, wherein thecompound capable of specific binding to the relaxin receptor (RXFP1) iscontained in a pharmaceutical composition comprising human relaxin-2 inadmixture with a pharmaceutically acceptable adjuvant, carrier, diluentor excipient.
 4. The method of claim 1, wherein the compound capable ofspecific binding to the relaxin receptor (RXFP1) is selected from thegroup of human relaxins comprising human relaxin-1, human relaxin-2,human relaxin-3 and biologically active analogues or derivativesthereof.
 5. The method of claim 1, wherein the compound capable ofspecific binding to the relaxin receptor (RXFP1) is contained in apharmaceutical composition formulated as an emulsion (oil-in-water orwater-in-oil) for delivery to the oral or gastrointestinal tract mucosa.6. The method of claim 1, wherein the compound capable of specificbinding to the relaxin receptor (RXFP1) is contained in a pharmaceuticalcomposition wherein a mucoadhesive protein is associated with thedelivery vehicle via a chemical or physical bond, so that thecomposition adsorbs to the mucosa or is retained on a mucosal surfacefor effecting systemic delivery of the relaxin.
 7. The method of claim1, wherein the compound capable of specific binding to the relaxinreceptor (RXFP1) is contained in a pharmaceutical composition forsubcutaneous infusion and administration of human relaxin-2 at a rate inthe range of 10 μg/kg/day to 1000 μg/kg/day.
 8. The method of claim 1,wherein H2 relaxin is administered at an infusion rate in the range of30 μg/kg/day to 100 μg/kg/day.
 9. The method of claim 1, wherein thepatient has been diagnosed suffering from a potential phosphorylationdeficit of the cardiospecific titin.
 10. The method of claim 1, whereinthe patient is further renally impaired.
 11. The method of claim 1,wherein the patient has a creatinine clearance in the range of 30 to 75mL/min/1.73 m² estimated according to the MDRD formula.
 12. The methodof claim 1, wherein the patient is hypertensive and/or suffering fromdiabetes and/or oxidative stress and/or inflammation.